Project Summary/Abstract B cell selection in germinal centers (GCs) is important for the success of antibody responses against influenza, HIV-1 and many other pathogens. However, the full set of signals involved in mounting GC responses is not understood. Recent work highlights the importance of signals from T follicular helper (Tfh) cells in driving preferential outgrowth of higher affinity B cells. The factors determining the strength and quality of signaling between Tfh cells and B cells are incompletely defined and are a focus of this proposal. The surface receptor HVEM (TNFRSF14) is frequently mutated in human GC-derived lymphomas (follicular lymphoma and GCB-diffuse large B cell lymphoma). This finding suggests HVEM has an important role in normal GC function but this role has not been defined. We have found that HVEM deficiency gives B cells a competitive advantage over wild-type B cells during GC responses. Our preliminary data suggest that this advantage only occurs when T cells are capable of sensing HVEM through expression of BTLA. We therefore propose a model where HVEM engagement of BTLA on Tfh cells restrains the amount of T cell help delivered to the B cell. The first Aim will define how HVEM restrains the competitiveness of B cells for representation in the GC. Adoptive transfer and conditional deletion approaches will be used to determine the stage(s) of the B cell response at which HVEM acts. The function of the HVEM intracellular domain will be tested using retroviral gene transduction approaches and the effects of HVEM deficiency on plasma cell formation, affinity maturation and recall responses will be determined. The second Aim will define how BTLA within CD4 T cells determines the quality of help received by the B cell. We will use retroviral gene transduction to test the role of the BTLA cytoplasmic domain in Tfh cell function. Analysis of T cells lacking the tyrosine phosphatases, SHP1 and SHP2, will define their roles in BTLA function. The influence of HVEM-BTLA engagement on cell-cell contact and T cell signaling will be studied using intravital 2-photon microscopy combined with the Salsa6f calcium reporter. Erk signaling will be followed using a new nuclear-cytoplasmic translocation reporter mouse line. The final Aim will examine how loss of the HVEM-BTLA interaction contributes to GC-derived lymphomagenesis. We will test whether BTLA functions as an extrinsic tumor suppressor by testing for accelerated outgrowth of Bcl2-overexpressing GC B cells in mice lacking BTLA in T cells. We will test for possible co-oncogenic effects of combined HVEM- and Gna13-deficiency. By defining how HVEM and BTLA act to control T cell help of B cell responses, this work may point to new approaches for augmenting B cell diversification and affinity maturation in GCs. An understanding of how HVEM constrains GC B cell accumulation, including cells over-expressing Bcl2, will provide insights that may lead to new approaches for treatment of FL and GCB-DLBCL.